Browsing by Subject "Viruses -- Reproduction"
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Item Inhibition of virus replication by cocaine: alterations in interferon production and calcium regulation(Texas Tech University, 1999-12) Grattendick, Kenneth JohnPrevious studies have demonstrated cocaine to have a myriad of effects on the immune system. Depending on which component of the immune system is studied and the methods used, cocaine has been shown to possess both stimulatory and inhibitory effects. Cocaine has also been associated with an increase in the risk of developing infectious diseases, although a direct correlation has yet to be determined. The studies presented in this dissertation describe the effects of cocaine on host resistance to viral infections. Cocaine was found to possess antiviral properties in macrophages (Mø). L929 cells, and Madin Darby canine kidney (MDCK) cells in vitro. This effect was dose-dependent in all of these cells with 100 µg/ml exhibiting the maximal inhibition. A similar dose-dependent inhibition was observed in M0 exposed to norcocaine, a metabolite of cocaine. However, other metabolites did not show any effects on virus replication. Cocaine induced a time-dependent increase in the antiviral activity of Mǿ that was not reproduced in L929 or MDCK cells. On average, the antiviral effect of cocaine required approximately 24—48 hr to appear, indicating that the effect was not due to a direct interaction of cocaine and virus. Cocaine's inhibition of virus replication could be reversed by the addition of either antibodies to interferons (IFN) or calcium ionophores. An indication that the effect of cocaine was due to the secretion of an antiviral protein was found in experiments showing that the antiviral effect could be transferred from cells incubated with cocaine to unstimulated cells. This antiviral product was found to accumulate over time in the media and could be neutralized by the addition of anti-IFN. In studies to determine the direct effects of cocaine on IFN production, cocaine was found to induce a 2-3-fold increase in IFN secretion in both L929 cells and M0, with similar increases in IFN transcripts. Cocaine also demonstrated the ability to inhibit cell proliferation, an effect attributed to the production of IFN. Experiments were also conducted to determine if the antiviral effects of cocaine observed in vitro could be demonstrated in an animal model. C57B1/6 mice were infected with Influenzavirus A and given daily i.p. injections of 10 mg/kg cocaine or saline. Cocaine-injected mice were visibly less sick than control animals and had 50% less virus in their lungs, as determined by hemagglutination. This reduction in virus load was consistent with the previous in vitro experiments with cocaine. Because the addition of calcium ionophores had been demonstrated to reverse the antiviral effect of/« vitro cocaine exposure, studies were conducted to determine the effects of cocaine on intracellular calcium (Ca^2+) regulation. Mø incubated with 100 µg/ml cocaine for 48-72 hr demonstrated a 41% increase in steady-state Ca^2+ concentrations. This effect was observed when cells were assayed either in the presence or absence of extracellular Ca^2+ indicating an alteration in calcium regulation that was localized to either the cytoplasm or intracellular membranes. Studies on the mobilization of Ca^2+ showed an increase m ATP-induced Ca^2+ transients when Mø were incubated with cocaine. Calcium ATPase inhibitors reduced the calcium increases in cocaine-treated Mø, further supporting the hypothesis that cocaine was increasing calcium mobilization. This result also indicated that the mechanism by which cocaine altered intracellular Ca levels was most likely localized to the endoplasmic reticulum Ca^2+ -ATPases. In summary, cocaine was found to inhibit virus replication by increasing IFN production and altering cytoplasmic Ca^2+ levels.Item Studies with semi-permissive infections of nuclear polyhedrosis viruses: evidence for intracellular restriction of host-range(Texas Tech University, 1983-08) Carpenter, William MichaelNuclear polyhedrosis viruses (Family: Baculoviridae) show great promise in agricultural pest control applications. However, in order for these viruses to be used most effectively, basic questions regarding the molecular basis of their replication, evolution, and host-specificity must be addressed. This thesis is concerned with delineating mechanisms of nuclear polyhedrosis virus host-specificity. An in vitro model was used to compare the importance of membrane-level and intracellular host-range mechanisms for nuclear polyhedrosis viruses. This model was composed of two systems: Autographa califomica NPV (Acal NPV) , strain Gl (Gerwig, 1981), which at low multiplicity produces semi-permissive infections in Spodoptera frugiperda cells, formed system I of the model. System II was composed of semi-permissive, S_. frugiperda NPV (SF NPV) infections in Trichoplusia ni cells. Transfection studies with Acal NPV Gl (System 1) showed that naked DNA does not enhance this semi-permissive infection. In addition, electron microscopy revealed that 50 percent of these infections produced progeny virus, but not polyhedral inclusion bodies, indicating that there is a host-range block in this system between virion morphogenesis and PIB formation. Infection kinetic studies with system II revealed that progeny virus was produced, but the yield was 700-fold less than positive controls. However, electron microscopy showed that the progeny virus was not enveloped, and was found in only five percent of infected TN cells; the remaining 95 percent of the cells contained forms of cytopathic effect such as virogenic stroma, but no virions. This suggests a host-range block at, or prior to, virion morphogenesis. These data strongly indicate intracellular, and perhaps intranuclear sites of host-range restriction for the above model infection systems. Further experiments based on these data should eventually lead to a better understanding of the genetic and biochemical mechanisms of host-range systems for the NPV. Once these mechanisms are understood, highly effective and specific strains of these viruses can be manufactured for the purpose of controlling agriculturally important pests.Item Studies with Semi-Permissive Infections of Nuclear Polyhedrosis Viruses: Evidence for Intracellular Restriction of Host-Range(Texas Tech University, 1983-08) Carpenter, William MichaelNuclear polyhedrosis viruses (Family: Baculoviridae) show great promise in agricultural pest control applications. However, in order for these viruses to be used most effectively, basic questions regarding the molecular basis of their replication, evolution, and host-specificity must be addressed. This thesis is concerned with delineating mechanisms of nuclear polyhedrosis virus host-specificity. An 2 ^ vitro model was used to compare the importance of membrane-level and intracellular host-range mechanisms for nuclear polyhedrosis viruses. This model was composed of two systems: Autographa califomica NPV (Acal NPV) , strain Gl (Gerwig, 1981), which at low multiplicity produces semi-permissive infections in Spodoptera frugiperda cells, formed system I of the model. System II was composed of semi-permissive, infections in. frugiperda NPV (SF NPV) infections in Trichoplusia ni cells. Transfection studies with Acal NPV Gl (System 1) showed that naked DNA does not enhance this semi-permissive infection. In addition, electron microscopy revealed that 50 percent of these infections produced progeny virus, but not polyhedral inclusion bodies, indicating that there is a host-range block in this system between virion morphogenesis and PIE formation. Infection kinetic studies with system II revealed that progeny virus was produced, but the yield was 700-fold less than positive controls. However, electron microscopy showed that the progeny virus was not enveloped, and was found in only five percent of infected TN cells; the remaining 95 percent of the cells contained forms of cytopathic effect such as virogenic stroma, but no virions. This suggests a host-range block at, or prior to, virion morphogenesis. These data strongly indicate intracellular, and perhaps intranuclear sites of host-range restriction for the above model infection systems. Further experiments based on these data should eventually lead to a better understanding of the genetic and biochemical mechanisms of host-range systems for the NPV. Once these mechanisms are understood, highly effective and specific strains of these viruses can be manufactured for the purpose of controlling agriculturally important pests.